RU2610208C1 - Ionic liquids as antimicrobial preparations - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention is intended for treatment of infectious processes, caused by sensitive microorganisms. The invention discloses application of N-decylpyridinium tetrachloroferrate as antimicrobial preparation.

EFFECT: application of the said compound, possessing antimicrobial action, ensures treatment of infectious processes, caused by sensitive microorganisms.

1 dwg, 1 tbl

Description

The invention relates to medicine and can be used in medical practice as an antimicrobial agent for the treatment of infectious processes caused by susceptible microorganisms.

Quaternary ammonium compounds containing one or two higher radicals in a molecule have been used in medical practice as antimicrobial agents for many years. A number of important regularities of the relationship between the chemical structure of these substances and their antimicrobial action have been established, which made it possible to create a number of effective drugs [A.G. Golikov, P.V. Reshetov, A.P. Krivenko et al., Chem.-farm. Zh., 39 (9), 23-25 (2005) .; A.A. Fefelov, Abstract. dis. Cand. Chem. Sciences, UFA (2006) .; O.V. Hutz, Pharmacist, 6, 1-10 (1998)]. The need for such preparations is determined, first of all, by the pronounced variability of microorganisms, the formation and wide distribution of their resistant variants, a general violation of ecological ties and intense environmental pollution [N.V. Klimova, H.M. Zaitseva, N.I. Avdyunina and others, Chem.-farm. Zh., 24 (1), 26-29 (1990)].

It is known that derivatives of quaternary ammonium and heterocyclic amines can be synthesized relatively easily. Of significant interest is the study of the mechanism of the antimicrobial action of these compounds at the cellular, subcellular and molecular levels, which will allow us to predict the spectrum of biological properties and to carry out directed synthesis of new compounds.

There is no doubt that the biological activity of quaternary amine salts is influenced by both the nature of the cation and the anion, therefore, the replacement of traditional halide anions in the salt structure with bulk types [PF ₆ ] ^- , [BF ₄ ] ^- , [FeCl ₄ ] ^- and other anions should affect the change in their biological activity. Such compounds - quaternary salts of ammonium, pyridinium, imidazolium, containing bulk hydrophobic or hydrophilic anions and located in a liquid state in a wide temperature range, called ionic liquids (IL), have recently attracted the attention of researchers in view of the uniqueness of their properties. Incombustibility, negligible vapor pressure and the hydrophobicity of IL practically exclude their ingress into the environment; non-toxicity determines the belonging of IL to the class of solvents that meet modern environmental requirements [P. Wasserscheid, T. Welton (eds.), Ionic Liquids in Synthesis, Wiley - VCH Verlag GmbH & Co. KgaA, Weinheim (2002), p. 355., H.V. Schweden, D.V. Chernyshev, I.V. Pletnev, Journal. Grew up. Chem. about them DI. Mendeleev, 42 (2), 80-91 (2008)].

Since the problem of the search for new antiseptic drugs remains relevant, it was interesting to trace whether antimicrobial activity persists for the corresponding ionic liquids obtained on the basis of the classical quaternary salts of ammonium, pyridinium, and morpholinium.

The following several works can serve as analogues. It is known to use as a antimicrobial agent a wide range of compounds belonging to the class of ionic liquids (US 20110144079 antimicrobial system). The disadvantage is that of the entire huge list of proposed compounds, antimicrobial activity (zones of growth inhibition of microorganisms) was studied only for a few individual compounds with a 1,3-dialkylimidazolium cation and chloride anion.

It is also known to use 2- (1'-hydroxy-4'-isopropenyl-1'-methylcyclohexyl-2-thio) -methylethanoate as an antimicrobial preparation with antimicrobial activity (RU 2556509). The disadvantage of this compound is the rather complicated method of its synthesis and ultimately the high cost.

It is known to use ionic liquids with a quinolinium and isoquinolinium cation with various anions as compounds having antimicrobial activity (RU 2010146005). The disadvantage is the use of additional compounds that maintain the viscosity, as well as the presence of metal ions: silver, copper, etc.

A patent may be used as a prototype (WO 2009125222 Antimicrobial system). The authors proposed to use a wide range of ionic liquids with aliphatic and heterocyclic nitrogen-containing cations and various organic and inorganic anions as antimicrobial agents. A disadvantage may be the lack of data on the antimicrobial activity of compounds containing pyridinium cation with a long chain radical and inorganic anions.

The antimicrobial properties of the claimed compounds: tetrafluoroborate, sulfate, nitrite, nitrate and tetrachloroferrate N-decylpyridinium are not described in the literature.

The aim of the invention is the search for effective compounds with increased antimicrobial activity.

This problem is solved by using the following compounds as antimicrobial agents: tetrafluoroborate, sulfate, nitrite, nitrate and N-decylpyridinium tetrachloroferrate, which has antimicrobial activity (Fig. 1).

The technical solution is to increase the effectiveness of the impact on the viability of various microorganisms.

The invention is illustrated in the drawing and table, which depict the following.

In FIG. 1. presents compounds with antimicrobial activity, where 1 is N-decylpyridinium nitrate, 2 is N-decylpyridinium sulfate, 3 is N-decylpyridinium terafluoroborate, 4 is N-decylpyridinium nitrite, 5 is N-decylpyridinium tetrachloroferrate.

Table 1 presents the antimicrobial activity of the studied compounds in relation to test cultures of pathogenic and conditionally pathogenic microorganisms.

The antimicrobial activity of the compounds was studied by the disk diffusion method (DDM) in agar on the lawn of test cultures: gram-positive Bacillus subtilis 6633, Staphylococcus aureus P209 ATCC 25923, Streptococcus pyogenes NCTC / 2696 / ATCC / 96/5; gram-negative E. coli ATCC 25922, Shigella sonnei III No. 1908, Salmonella typhimurium 5715, Pseudomonas aeruginosa ATCC 27853 and yeast Candida albicans ATCC 885-653. The antibacterial activity of the material was expressed in mm of growth inhibition diameter. High activity - diameter of the zone of growth inhibition of more than 25 mm; active - the diameter of the zone of growth inhibition is 16-25 mm; low activity - the diameter of the zone of growth inhibition of 10-15 mm

To determine the sensitivity of DDM, Mueller-Hinton agar (for streptococci with the addition of mutton blood) was used, prepared in accordance with the manufacturer's instructions. The thickness of the agar layer in the cup was 4.0 ± 0.5 mm, which was achieved when strictly 20 ml of agar was introduced into a Petri dish with a diameter of 90 mm and 25 ml of agar with a diameter of 100 mm. Before filling with molten medium, Petri dishes were placed on a strictly horizontal surface (level-adjusted, without depressions and bulges). After filling, the plates were left at room temperature to solidify. Freshly prepared plates were used, which were dried before inoculation by incubation at 37 ° С with the lid ajar for 10-20 min. Before inoculation, the absence of liquid condensate on the inner surface of the caps was monitored.

To determine the sensitivity of DDM, we used standardized ND-PMP-1 discs made of technical filter paper GOST 6722-75 (Federal State Institution for Supervision of Consumer Rights Protection and Well-being of St. Petersburg Scientific and Research Institute of Epidemiology and Microbiology L. Pasteur) person).

Storage of ND-PMP-1 disks from technical filter paperboard was carried out in sealed packaging of the manufacturer in a dry, dark place at a temperature of 2-8 ° C for the entire shelf life of the kit. Small batches of disks used during everyday work were stored tightly corked at temperatures up to 25 ° С for no more than 15 days so as to ensure that moisture could not get into the vial, in addition, for additional protection against moisture, special desiccant is contained in vials (cartridges) with disks (silica gel). Before use, the vials with disks were kept at room temperature 18-25 ° C for 1 h to prevent condensation on the inner wall of the vial. An opened bottle with disks was stored at a temperature of 2-8 ° C for the entire shelf life of the kit, provided that the color of the indicator silica gel was kept from light blue to blue. Immediately before use, the discs were impregnated with the test compound. As a control, disks saturated with distilled water were used.

To determine the sensitivity of DDM, a standard inoculum corresponding to a density of 0.5 according to the McFarland standard and containing approximately 1.5 × 10 ⁸ CFU / ml was used. Inoculum was used for 15 minutes after preparation. For inoculation of prepared plates with agar, sterile cotton swabs of industrial production were used (stick-swab (plastic-cotton) CITOSWAB sterile in individual packaging). The tampon was immersed in a standard suspension of the microorganism, then the excess inoculum was removed by squeezing the tampon against the walls of the tube. Inoculation was performed by dashed movements in three directions, turning the Petri dish 60 °.

Not later than 15 min after inoculation, disks impregnated with the studied compounds and distilled water were applied to the surface of the nutrient medium. The application of the discs was carried out using sterile tweezers. The distance from the disk to the edge of the cup and between the disks was 15–20 mm. For uniform contact with the surface of the agar discs were gently pressed with tweezers.

Immediately after the discs were applied to the surface of the nutrient medium, Petri dishes were placed upside down in the thermostat and incubated at 37 ° C for 18-24 hours (depending on the type of microorganism being tested).

After incubation, the plates were placed upside down on a dark matte surface so that light incident on them at an angle of 45 ° (taking into account reflected light). The diameter of the growth inhibition zones was measured with an accuracy of 1 mm using a ruler (Hi Antibiotic Zone Scale (PW 2097) (Hi Media Laboratories Pvt. Limited, India)).

The antimicrobial preparation dioxidin (a derivative of di-N-hydroxyquinoxaline), widely used in medical practice, was used as a comparison drug. This drug has high in vitvo chemotherapeutic activity on model infections similar in pathogenesis to human pathological processes (purulent meningitis, pyelonephritis, septicopyemia) and caused by strains of anaerobic bacteria that are resistant (including multiresistant) to drugs of other classes, including Pseudomonas aeruginosa strains. Dioxidin is characterized by a wide antibacterial spectrum with a bactericidal effect, and is also active against gram-positive and gram-negative aerobic conditionally pathogenic bacteria. The activity of dioxidine against mycobacterium tuberculosis is shown (E. Padeiskaya. Antibacterial preparation dioxidine: features of biological action and significance in the treatment of various forms of purulent infection / E. N. Padeiskaya // Infections and antimicrobial therapy. - 2011. - T. 3 - No. 5. - S. 105-155).

The antimicrobial activity of the compounds was expressed in mm of diameter growth retardation. The results of the determination are presented in Table 1.

All compounds showed medium and high antimicrobial activity against all test cultures of microorganisms.

The samples showed the most pronounced activity against pyogenic streptococcus (Streptococcus pyogenes), growth retardation zones ranged from 20 to 31 mm in diameter, and spore-forming hay bacillus (Bacillus subtilis), the diameter of the growth retardation zone was 20-26 mm. In relation to another gram-positive microorganism Staphylococcus aureus (Staphylococcus aureus), samples No. 2-5 and medium No. 15-18 (15–18 mm) possessed high bacteriostatic activity (20–22 mm).

Gram-negative microorganisms (E. coli, Shigella, Salmonella, Pseudomonas) showed an intermediate sensitivity to the studied compounds. The diameter of the growth inhibition zone was 10-19 mm. The highest antimicrobial activity in relation to the vast majority of microorganisms was shown by N-decylpyridinium tetrachloroferrate, and its activity against strains of microorganisms E. coli, Shigella sonnei, Candida albicans, Bacillus subtilis is higher than that of the reference drug - dioxidine. All tested compounds showed antimicrobial activity at the level of dioxidine or higher against most gram-positive and gram-negative microorganisms.

Candida albicans yeast showed intermediate sensitivity to the studied compounds with the same growth retardation zone of 15 mm in diameter, except for compound No. 4, which was highly active.

Thus, all the studied compounds have a wide spectrum of antibacterial activity. Moreover, to a greater extent, these samples act on gram-positive bacteria streptococci and staphylococci, which cause mainly pyogenic infections, as well as pneumonia, rheumatism, scarlet fever, erysipelas, colitis and other diseases of the skin, mucous membrane, intestines and other organs, including caries teeth, and can be offered as antimicrobials.

The average antimicrobial activity of the compound was exerted on gram-negative microorganisms of the intestinal group of bacteria (Escherichia, Shigella, Salmonella, Pseudomonas), causative agents of Escherichiosis, bacterial dysentery, Salmonella and on yeast fungi of the genus Candida.

Claims (1)

The use of N-decylpyridinium tetrachloroferrate as an antimicrobial agent.

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